dienelactone and 4-chlorocatechol

Pseudomonas aeruginosa RW41 is the first bacterial strain, which could be isolated by virtue of its capability to mineralize 4-chlorobenzenesulfonic acid (4CBSA), the major polar by-product of the chemical synthesis of 1,1,1-trichloro-2,2-bis-(4-chlorophenyl)ethane (DDT). This capability makes the isolate a promising candidate for the development of bioremediation technologies. The bacterial mineralization of 4CBSA proceeds under oxygenolytic desulfonation and transient accumulation of sulfite which then is oxidized to sulfate. High enzyme activities for the turnover of 4-chlorocatechol were measured. The further catabolism proceeded through 3-chloromuconate and, probably, the instable 4-chloromuconolactone, which is directly hydrolyzed to maleylacetate. Detectable levels of maleylacetate reductase were only present when cells were grown with 4CBSA. When the ordinary catechol pathway was induced during growth on benzenesulfonate, catechol was ortho-cleaved to cis,cis-muconate and a partially purified muconate cycloisomerase transformed it to muconolactone in vitro. The same enzyme transformed 3-chloro-cis,cis-muconate into cis-dienelactone (76%) and the antibiotically active protoanemonin (24%). These observations are indicative for a not yet highly evolved catabolism for halogenated substrates by bacterial isolates from environmental samples which, on the other hand, are able to productively recycle sulfur and chloride ions from synthetic haloorganosulfonates.

Topics: Benzenesulfonates; Catechols; Chromatography, Ion Exchange; DDT; Furans; Hydrocarbons, Halogenated; Intramolecular Lyases; Lactones; Maleates; Metabolic Networks and Pathways; Oxidation-Reduction; Pseudomonas aeruginosa; Sorbic Acid; Sulfates; Sulfites

Ralstonia eutropha JMP134 (pJP4) harbors two functional gene clusters for the degradation of chlorocatechols, i.e. tfdCDEF (in short: tfd (I)) and tfdD (II) C (II) E (II) F (II) (in short: tfd (II)), which are both present on the catabolic plasmid pJP4. In this study, we compared the function of both gene clusters for degradation of chlorocatechols by constructing isolated and hybrid tfd (I)- tfd (II) clusters on plasmids in R. eutropha, by activity assays of Tfd enzymes, and by HPLC/MS of individual enzymatic catalytic steps in chlorocatechol conversion. R. eutropha containing the tfd (II) cluster alone or hybrid tfd-clusters with tfdD (II) as sole gene for chloromuconate cycloisomerase were impaired in growth on 3-chlorobenzoate, in contrast to R. eutrophaharboring the complete tfd (I) cluster. Enzyme activities for TfdD(II) and for TfdE(II) were very low in R. eutropha when induced with 3-chlorobenzoate. By contrast, a relatively high enzyme activity was found for TfdF(II). Spectral conversion assays with extracts from R. eutropha strains expressing tfdD (II) all showed accumulation of a compound with a similar UV spectrum as 2-chloro- cis,cis-muconate from 3-chlorocatechol. HPLC analysis of in vitro assays in which each individual step in 3-chlorocatechol conversion was reproduced by sequentially adding cell extracts of an Escherichia coli expressing one Tfd enzyme only demonstrated that TfdD(II) was unable to cause conversion of 2-chloro- cis,cis-muconate. No accumulation of intermediates was observed with 4-chlorocatechol. From these results, we conclude that at least TfdD(II) is a bottleneck in conversion of 3-chlorocatechol and, therefore, in efficient metabolism of 3-chlorobenzoate. This study showed the subtle functional and expression differences between similar enzymes of the tfd-encoded pathway and demonstrated that extreme care has to be taken when inferring functionality from sequence data alone.

Topics: Adipates; Amino Acid Sequence; Base Sequence; Catechols; Chlorobenzoates; Cloning, Molecular; Cupriavidus necator; Escherichia coli; Intramolecular Lyases; Lactones; Molecular Sequence Data; Plasmids; Sorbic Acid